Data presented in this report proved that codon optimization was effective for improving the antigen expression and immunogenicity of flu HA DNA vaccines. In early flu DNA vaccine studies, there were only low or undetectable HA-specific antibody responses after DNA immunizations. Positive antibody responses were detected only after a flu virus challenge (9
). In the last several years, codon optimization has been shown effective for improving the immunogenicity of DNA vaccines expressing human immunodeficiency virus antigens (1
). In addition, DNA vaccines with codon-optimized antigen gene inserts have been used successfully against viral (such as severe acute respiratory syndrome) (53
), papillomaviral (3
), bacterial (21
), and parasitic pathogens (17
). By using an H1 serotype HA antigen as the model system in the current study, we compared the relative levels of antigen expression and immunogenicity between wild-type and codon-optimized flu HA DNA vaccines in two animal models (mouse and rabbit) with two DNA vaccine delivery methods (gene gun and intramuscular needle injection). The data clearly demonstrated that the codon-optimized HA DNA vaccines were able to induce better HA-specific antibody responses than were the wild-type gene sequences because codon-optimized HA DNA vaccine was able to induce a quicker rise and higher peak-level anti-HA antibody responses.
The ability of eliciting high-titer, anti-HA antibodies by DNA immunization prior to the viral challenge allowed us to expand our study to further measure the levels of protective antibody components in animal sera immunized with HA antigens from either H1 or H3 serotypes. Both high-level HI and neutralizing activities were easily elicited by codon-optimized DNA vaccines. The titers of protective antibodies achieved in this report were not only significantly improved over that from early versions of flu DNA vaccines (2
) but also comparable to or higher than those of positive anti-HA sera reported in previous animal studies with various flu vaccines, including recombinant viral vector-based flu HA vaccines (16
) and inactivated and live flu vaccines (23
It is very interesting to discover that the protective antibody responses were dependent on the forms of HA antigen inserts. For the H1 HA antigen used in the current study, only the full-length form was able to induce high levels of protective antibodies, while both the full-length and the TM-truncated H1 HA antigens elicited very similar levels of HA-specific binding antibody responses. Therefore the full-length H1 HA antigen was more effective for preserving certain protective antigen conformation than the TM-truncated form of H1 HA antigen was. On the other hand, both the full-length and the TM-truncated forms of H3 HA antigens were able to elicit similar levels of protective antibodies. Singh et al. reported that the truncated anchor-free HA could form monomers or trimers depending on the strain and construct used (44
). It is likely that the truncated H1 HA DNA vaccine produces the monomeric forms, and truncated H3 HA DNA vaccine produces the trimeric forms, which would contribute to the differences in conformation-sensitive protective antibody responses.
Consistent with the above finding that H1 serotype HA and H3 serotype HA antigens are conformationally different, we found that the H1 HA antigen was resistant to natural cleavage into its two subunits, while the H3 HA antigen was easily detected in the forms of HA1 and HA2 subunits in transiently transfected 293T cells. Treanor et al. conducted a recent vaccine study in humans using a trivalent, baculovirus-expressed, recombinant HA vaccine based on the HAs of influenza A/Panama/2007/99 (H3N2), A/New Caledonia/20/99 (H1N1), and B/Hong Kong/330/2001 (52
) and concluded that the H3 component, but not the H1 and influenza virus type B components, elicited a robust neutralizing antibody response in humans. Although the authors could not explain the reasons for these differences, their results are in agreement with our study, in which we found that H1 HA-based vaccines are less immunogenic when lacking the appropriate transmembrane and cytoplasmic domains.
Our results will have an important impact on the selection and design of HA antigens for developing the most protective flu vaccines. Traditionally, in order to study the structure requirement for immunogenicity and protection by individual HA antigens, the flu viral stocks expressing different HA antigens in their native or modified forms would have to be produced, inactivated, concentrated, and purified. This process was shown in a recent effort studying the role of specific HA amino acids in the immunogenicity and protection of H5N1 influenza virus vaccines (13
). Such a process is complicated and may introduce multiple variables to the final immunization studies. There has been a limited effort to produce HA antigens by a recombinant protein approach, presumably due to the challenges of cost and time when facing the large number of HA antigens existing in the world, as the results of antigen drift, even within one serotype of flu viruses. The discovery of DNA immunization technology in the early 1990s initially led to high enthusiasm that this novel approach may be a simple alternative to the traditional flu vaccines. However, the low immunogenicity of early DNA vaccine designs, especially in higher animal species or humans when delivered without the help of physical delivery methods (gene gun or electroporation), quickly dampened such hope. One important early observation was that flu DNA vaccines were unable to elicit high-level, HA-specific antibody responses prior to viral challenge (9
). In the last decade, significant improvement has been made in DNA vaccination approaches. However, such improvement has not been well incorporated into flu vaccine applications. Data presented in this report confirmed that codon optimization was effective for improving the immunogenicity of HA DNA vaccines. With a codon-optimized DNA vaccination approach, including the use of the gene gun delivery method, we report here that it is feasible to reliably elicit high-level protective antibody responses against flu HA antigens. This will provide a powerful tool to greatly accelerate the identification, selection, and rational design of the most protective HA antigens, which can then be incorporated into either conventional or novel flu vaccine strategies in either wild-type or codon-optimized gene sequences.
The development of high-immunogenicity HA DNA vaccines also provides an alternative approach to the development of vaccines against pandemic influenza caused by avian flu viruses since these emerging flu viruses do not grow well enough to produce high-yield viral stocks for the manufacturing of inactivated vaccines. Recent HA DNA vaccine studies in humans have demonstrated that significant levels of HA-specific antibody responses are induced when such DNA vaccines are delivered by a gene gun (7
). With DNA vaccination, it is easy to mix several protective antigens in one delivery. Our data with HA antigens from both H1 and H3 serotypes confirmed that, relative to monovalent antigen formulation, such polyvalent formulations can be equally effective in eliciting protective antibody responses.